J. Med. Chem. 1993,36, 2300-2310
2300
Potent HIV Protease Inhibitors: The Development of Tetrahydrofuranylglycines as Novel Pz-Ligands and Pyrazine Amides as Ps-Ligands Arun K. Ghosh,’J Wayne J. Thompson,+M. Katharine Hollowayf Sean P. McKee,t Tien T. Duong,t Hee Yoon Lee,t Peter M. Munson,+Anthony M.Smith,? Jenny M. Wai,t Paul L. Darke,s Joan A. Zugay,# Emilio A. Emini,l William A. Schleif,* Joel R. Huff,’ and Paul S. Anderson+ Departments of Medicinal Chemistry, Molecular Biology, Molecular Systems, and Virw and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486 Received April 5,1993
A series of protease inhibitors bearing constrained unnatural amino acids a t the P2-position and novel heterocycles a t the P3-position of compound 1 (Ro 31-8959) were synthesized, and their in vitro enzyme inhibitory and antiviral activities were evaluated. Replacement of Pn-asparagine of compound 1with (2S,3’R)-tetrahydrofuranylglycineresulted in improvement in enzyme inhibitory as well as antiviral potencies (compound 23). Interestingly, incorporation of (2S,3’S)-tetrahydrofuranylglycine a t the Pz-position proved to be less effective. The resulting compound 24 was 100-foldless potent than the 2S,3R-isomer (compound 23). Thisstereochemical preference indicated a hydrogen-bonding interaction between the tetrahydrofuranyl oxygen and the residues of the &-region of the enzyme active site. Furthermore, replacement of P3-quinolinoyl ligand of 1with various novel heterocycles resulted in potent inhibitors of HIV proteases. Of particular interest, compound 2 with (2S,3’R)-tetrahydrofuranylglycine a t P2 and pyrazine derivative a t P3 is one of the most potent inhibitors of HIV-1 (ICmvalue 0.07 nM) and HIV-2 (I& value 0.18 nM) proteases. Another important result in this series is the identification of compound 27 in which the P2-P3amide carbonyl has been removed. The resulting compound 27 has exhibited improvement in antiviral potency while retaining the enzyme inhibitory potency similar to compound 1. The inhibition of the enzyme HIV-1 protease, which cleaves thegug andgag-pol polyproteinsinto the functional proteins of infectious virions, continues to be a major therapeutic target for the treatment of AIDS and related ailments.’ Of the numerous potent protease inhibitors that have been reported recently? the present clinical candidate (2S,4aS,BaS,2’R,3’S)-N-tert-butyl-2-(2’-hydroxy4’-phenyl-3’-((N-(2-quinolinylcarbonyl)-~-asparaginyl)amino)butyl)decahydroisoquinoline-3-carboxamide(1) (Ro 31-8959) is particularly unique because of its effectiveness against both the enzymes HIV-1 and HIV-2 protease^.^ Since these are the genetically most divergent strains of HIV known to exist to date, a protease inhibitor with this property may result in reduced susceptibility to clinical resistance. Subsequently, we have investigated the effect of incorporationof conformationally constrained unnatural amino acids in place of asparagine at the P2 subsite of compound 1. As reported in a recent paper: the replacement of asparaginewith (2S,3’R)-tetrahydrofuranylglycine not only increased the enzyme affinities for HIV-1 and HIV-2 proteases but led to significant enhancement of antiviral potencies compared to 1. We now report the synthesis, enzyme inhibition, and antiviral potencies of a structurally novel class of protease inhibitors in which various constrained unnatural amino acids and novel heterocyclic derivatives were incorporated at the P2- and Ps-positions of the present clinical candidate 1 (Ro 318959). This study has resulted in protease inhibitors with improved enzyme inhibitory and antiviral potencies. Particularly noteworthy is the inhibitor 2 which is very potent against HIV-1 (ICm = 0.07 nM) and HIV-2 (ICm = 0.18 nM). t Department of
Medicinal Chemistry. Molecular System. Molecular Biology. 1 Department of V i m and Cell Biology.
t Department of 1 Department of
Scheme I. Synthesis of Azido Epoxidea
3
Ph’
‘
6
+ 5
Id.C 0 N
3
A
Ph’
Key: (a) PhMgBr, CuCN, THF, -78
O C ; (b)tBuOOH, (-)-DET, Ti(0iPrk CHzClz, -22 O C ; (c) Ti(OiPr)a(Ns)z, PhH, 80 O C ; (d) AcOCMeZCOCl, CHCb, 23 O C ; (e) NaOMe, THF, 23 O C .
Chemistry The desired (!2R,3S)-azido epoxide 7 was prepared as shown in Scheme I. The commerciallyavailablebutadiene monoxide 3 was converted to allylic alcohol 4 by reaction with phenylmagnesium bromide in the presence of a catalytic amount of cuprous iodide. The allylic alcohol 4 was then subjected to the Sharplessepoxidationscondition with (-)-diethyl D-tartrate to furnish the epoxide 5. Regioselective ring opening of epoxide 5 with diisopropoxytitanium diazide in benzene at 75 “C,as described by Sharpless and cO-workers16afforded the azidodiol6in very good yield. Azidodiol6 was then converted efficiently to the desired azidoepoxide 7 by treatment with 2-acetox-
0022-26231931l836-23OO$O~.oOlO 0 1993 American Chemical Society
Potent HZV Protease Inhibitors
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16 2301
Scheme 11. Synthesis of Amino Acid'
Table I. Structure and Inhibitory Potencies of Various Constrained PrLiganda
n
8
t
Compd
1.
23.
I'
ICs0 (nM)
CICw (nM)
0.23M.1
22f7
(n=3)
(n=10)
0.054M.027
8f4
(n=4)
r 9
r 9
12
R
24.
5.4
25.
2.6
26.
0.7
31.
0.6
32.
3.3
33.
0.5
34.
39
35.
67.9
U
\Ph I1
r 1 3 X=N3 4 1 3 a X=NH2
Key: (a) MaC1, E t N , CH2C12, -10 OC; (b) NaH, CH2(C02Et)2, DMF;(c) 1 N NaOH then HsO+; (d) CunO (cat), CHsCN, 80 O C ; (e) MeCOCl, E t a , THF, -78 OC then ~-lithio-(s)-(-)-4benzy~olrazolidinone; (f) KN(TMS)a,THF, -78 "C, 30 min, then trisyl azide, -78 OC, 2 min, then AcOH, 30 "C, 1 h; (g) LiOH, THF-H20 (h) 6% Pd-C, EtOH-H20. a
yisobutyryl chloride in chloroform at 23 "C followed by exposure of the resulting chloroacetate derivative to an excess of sodium methoxide in tetrahydrofuran at 23 "C for 3 h.' The synthetic route leading to the (2S,3'R)-tetrahydrofuranylglycine equivalent is described in Scheme 11. Readily prepared* enantiomerically pure (S)-(+)-3-hydroxytetrahydrofuran (8) was mesylated with mesyl chloride and triethylamine in methylene chloride at 0 "C for 20 min. DisplacementBof the resulting mesylate with the sodium salt of diethyl malonate in DMF at 100 "C furnished the malonate derivative 9. Ester hydrolysis followed by copper ion promoted decarboxylationlO furnished the (R)-tetrahydrofuranylaceticacid (10) in very good yield. The highly diastereoselective azidation protocol developed by Evansll was then employed to introduce the a-amine functionality. Thus, deprotonation of the (S)-(-)-4-benzyloxazolidinone with n-BuLi followed by acylation with the mixed anhydride resulting from the reaction of acid 10 with pivaloyl chloride in the presence of triethylamine provided the carboximide 11 after silica gel chromatography. Treatment of this carboximide with potaseium hexamethyldisilazidein tetrahydrofuran at -78 "C for 30 min provided the potassium enolate which was reacted with trisyl azide at -78 O C for 2 min and then quenched with glacial acetic acid and warmed to 30 "C. The a-azido carboximide thus obtained was purified by silicagel chromatography to furnish the azide 12 as a single diastereomerby HPLC and 'H-NMR (400-MHz)analysis. Removal of the chiral auxiliary was effected by exposure to lithium hydroxide in aqueous tetrahydrofuran to provide the desired acid 13. The resulting azido acid was hydrogenated over 5% palladium on charcoal in a mixture of
ethanol and water (2:l) to furnish the amino acid 13a. The corresponding (2S,3'S)-tetrahydrofuranylglycine equivalent was obtained utilizing (R)-(-)-3-hydroxytetrahydrofuran as the starting material. Similarly, diastereomeric azido acid 28 and other cyclic amino acids utilized in Table I were prepared following a similar course of reaction as described in Scheme 11. Various pyrazine derivatives were synthesized as shown in Scheme 111. The known12 dichloropyrazine derivative 14 waa heated with dimethylamine or l-methyl piperazine in 2-propanol at 85 "C for 12 h to furnish the pyrazine derivative 1Sa or 1Sb. Hydrolysisof the correspondingmethyl ester with aqueous NaOH in ethanol and subsequent acidification provided the acid derivative. For the preparation of pyrazine derivative 17, pyrazine 1Sb was first converted to bromide
2302 Journal of Medicinal Chemistry, 1993, Vol. 36, No.16
Scheme 111. Synthesis of Pyrazine Derivatives0
Ghosh et al.
Scheme Va 7 0
C02Me
a 15a X = NMez 15b X = NANMe
CI
14
n
C02Me
CI
Key: (a) dimethylamine or l-methyl piperazine, iPrOH, 85 "C; (b)NaN02, Brz, HBr, AcOH; (c) Hz, 5%P d C , THF; (d) 1 N NaOH, EtOH then HsO+.
Scheme IVa H
Me' 2 Key: (a) quinaldic acid, PhZPOC1, EbN, THF, -10 "C,1 h, then 23 "C,3 h; (b) pyrazine 14, iPrOH, 84 "C,12 h; (c) acid 17, PhSOCl, EON, THF, -10 "C,2 h, then 23 "C, 3 h. 7
18
H
21 X = N 3 dL22
H
20
X=NH2
Key: (a) iPrOH, 80 "C,12 h; (b)Ha, 10%Pd-C, AcOH, MeOHTHF, 12 h; (c) azido acid 13, EDC, HOBt, EtaN, DMF; (d) Hz, 5% P d C , AcOH, MeOH-THF, 12 h. 0
16 by reaction with bromine and sodium nitrite in a mixture of glacial acetic acid and 48 % hydrobromic acid. Bromo ester 16 was then debrominated upon hydrogenation over 10% palladium on charcoal, and the resulting ester was saponified to furnish acid 17. Various hydroxyethylamine isosteres containing tetrahydrofuranylglycine at the P2 position were prepared following the general synthetic route outlined in Scheme IV. Azido epoxide 7 and decahydroisoquinoline13J4derivative 18 were heated at reflux in 2-propanol for 12 h to furnish the azido alcohol 19 in 83% yield after silica gel chromatography. Catalytic hydrogenation of azide 19 with 10% palladium on charcoal in a mixture of tetrahydrofuran and methanol (41) in the presence of acetic acid afforded the amine 20 in excellent yield. Using a standard peptide couplingprocedure,l6amine 20 was reacted with the azido acid 13 in the presence of N-ethyl-N'-(3-(dimethylamino)propy1)carbodiimide hydrochloride, triethylamine, and 1-hydroxybenzotriazole hydrate in DMF to furnish the azide 21 which was hydrogenated over 5 % palladium on charcoal to provide amine 22. The amine 22 was then converted to various protease inhibitors by standard coupling reaction with the corresponding acid (Scheme
V). For example, reaction of quinaldic acid with diphenylphosphinic chloride and triethylamine in tetrahydrofuran at -10 O C for 1h followed by addition of amine 22 afforded the inhibitor 23 in excellent yield.16 Similarly, reaction of acid 17 with amine 22 resulted in the inhibitor 2. The N-pyrazinyl compound 27 has been prepared by reaction of pyrazine derivative 14 and the amine 22 in refluxing 2-propanol for 12 h. Various inhibitors with tetrahydrothiophene derivatives or sulfolanes at the P2 position were synthesized according to Scheme VI. Azido acids 28 were prepared as a mixture of diastereomerd following Evans' asymmetric azidation protocol described in Scheme 11. The couplingof acid 28 with amine 20 under standard conditions afforded the coupling products 29 and 30. The isomers were easily separated by column chromatography over silica gel, and the stereochemistry at the 3-position of the tetrahydrothiophene ring was assigned by comparison of lH NMR (300 MHz) spectra of the azide 21 and the diastereomer of 21 that contains the 2(S)-azido-3(S)-tetrahydrofuranylmoiety. The azide derivatives 29 and 30 were then converted to inhibitors 31 and 32 following a similar course of reaction as described for inhibitor 23. Further assignments of tetrahydrothiophene ring stereochemistry of compound 31 and 32 were made based on comparison of lH NMR of the correspondingtetrahydrofuranyl derived inhibitors 23 and 24. Selective oxidation" of the ring sulfur of inhibitors 31 and 32 with a catalytic amount of osmium tetraoxide and an excess of N-methylmorpholineN-oxide in a mixture (3:l) of acetone and water furnished the sulfolane derivatives 33 and 34 in good yield. Various inhibitors in Tables I and I1have been synthesized by following a similar course as shown in Schemes V and VI. Results and Discussion The X-ray crystal structure of the enzyme-inhibitor complex of L-689,502 bound to HIV-1 protease (2.25 A
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16 2303
Potent HIV Protease Inhibitors
Scheme VIe
n
7 X
Table 11. Structure and Inhibitory Potencies of Various Constrained Pa-Ligands H.. R-N-NH
Ph’ I
h
Compd
R
n
‘0
0
H
ICW(nM) CICe5 (nM)
0.071fl.OO3
12
(n=2)
a
+
H
0.16
3
0.39
-_-
0.12
12
2.83
-__
0.24
25
0.06
12
0
28
H
Ib, C T X
n
32X=S Key: (a) amine 20, EDC, HOBt, Et3N, DMF (b) H2,10%Pd-C, MeOH-EtOAc; (c) quinaldic acid, Ph2POC1, EtsN, THF, -10 OC, 1 h, then 23 “C,3 h; (d) OsO~,4-methylmorpholineN-oxide, acetonewater. a
resolution)18 was utilized to construct the modeled structure of compound 1 (Ro 31-8959). The structure was then energy minimized in the active site using the Merck molecular force field, OPTIMOL,lg which is a variant of the MM2 program.20 An examination of the modeled structure of inhibitor 1revealed that the carbonyl oxygen of the asparagine is within hydrogen bonding distance to the asp 29 and asp 30 NH present in the SZbinding domain of the HIV-1 protease. On the basis of this possible insight, we hypothesize that a conformationally constrained cyclic ether oxygen might donate ita loan pairs for hydrogen bonding to the appropriate residues in the SZregion of the enzyme active site. Such a design may effectively rep€ace the Pz-asparagine, providing an inhibitor with improved in vitro potencies and pharmacokinetic properties. Indeed, as can be seen from Table I, the replacement of the asparagine of compound L21 with (2S,3’R)-tetrahydrofuranylglycine (compound 23) resulted in an over 4-fold increase in ita inhibitorypotencymZ2 Interestingly, (2S,3’S)tetrahydrofuranylglycine(3’(S)-Thfg)containing inhibitor (compound 24) showed a 16-foldloss in potency compared to 1. Furthermore, examination of the cyclopentyl derivative 25 established that the ring oxygen is essential for potency enhancement. Also, as expected, the inhibitory potency of the corresponding acyclic analog 26 is over 17fold less potent than the compound 23. The fact that the 3’(R)-Thfg-derived inhibitor 23 is more potent than the 3’WThfg-derived inhibitor 24 suggested that 3’(R)-Thfg is successfullymimicking the enzyme-bound conformation of the asparagine side chain of compound 1. Accordingly,
0.15
25
0.11
25
the active model forthe inhibitor 23 was created and based on the superimposition on the model structure of 1 (Figure 2); it appeared that the tetrahydrofuran oxygen of 23 is in close proximity to the carbonyl oxygen of the asparagine m0iety.~3Thus, the position of the oxygen in 3’(R)-Thfg seems to set up for hydrogen-bonding interaction with the asp 29 and asp 30 of the HIV-1 protease. The antiviral potencies of the inhibitors 23 and 24 are consistent with their enzyme inhibitory potencies. As is evident in Table I, 3’(R)-Thfg-derivedinhibitor 23 has prevented the spread of HIV-1 in MT4 human T-lymphoid cells infected with IIIb isolatelsat an average concentration of 8 nM (CICes), a 3-fold potency enhancement over inhibitor 1. In contrast, 3’(S)-Thfg derived inhibitor 24 has exhibited an antiviral potency of 100 nM. The synthesis of compounds 31-35 was undertaken to evaluate the influence of other heteroatoms and heterocycles on the enzyme inhibitory and antiviralpotencies. Tetrahydrothiofuranylglycine-derived inhibitor 31 with a 3’R configuration is significantly less
2304 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16
= 0.4 nM = 0.5 nM
ICso [HIV-I] [HIV-2]
II 2 ICso [HIV-I] = 0.07nM [HIV-21 = 0.18 nM Figure 1.
potent (ICs0 0.6 nM) than the corresponding tetrahydrofuran derivative 23. Since the sulfur atom in the tetrahydrothiopheneringisa poor hydrogen bond acceptor,x a weak hydrogen-bondinginteraction may account for this loss of potency. The stereochemical preference for 3’Rconfiguration by this binding region, however, remained consistent with our earlier observation with the 3’-Thfgderived inhibitors. Next, in order to examine the effect of sulfone o m e n s in this binding pocket, the corresponding sulfolane derivatives were prepared. The sulfolane derivative with a 3%-configuration is equipotent to the corresponding sulfide (compound 33, IC% 0.5 nM). On the other hand, the sulfone derivative with a 3’8configuration turned out to he 10-fold less potent than the sulfide 32. Furthermore, the replacement of the 3-tetrahydrofuran ring in 23 with 4-tetrahydropyran (compound 35) was examined. This substitution has resulted in a dramatic loss in enzyme inhibitory potency (compound 35, ICw 68 nM). The reason for this loss of potency is probably due to the increase in ring size as well
Ghosh et al.
as the position of the oxygen, which impedes the access to the appropriate residues for specific interaction in the SZsubsite of HIV-1 protease. Thus,from these studies, it is quite apparent that 3’(R)-Thfg is a highly effective asparagine surrogate for the SZ region of the HIV-1 substrate binding site. In an attempt to further improve the in vitro potencies and pwiblythe pharmacokinetic properties of 3’(R)-Thfgderivedinhibitor23,wehaveincorporatedvariouspyrazine derivatives and heterocycles at the Pa position. As ahown in Table 11,substitution of the 2-quinolinoyl moiety in 26 with the quinoxalinoyl group resulted in (compound 36) a slight improvementin potency. However, incorporation of the 2-quinoxalinoylmoiety in 23 afforded the inhibitor 37 with an ICm value of 0.12 nM, a 2-fold loss in potency compared to 23. The removal of the second aromatic ring of the 2-quinoxalinoylmoiety in 37 provided unsubstituted pyrazine-derived inhibitor 38 (ICs0 2.8 nM) with a more than 20-fold loss in potency. Next, we have examined a number ofpyrazinederivativeswithvariousfunctionalities in order to determine whether or not substitution on the pyrazine ring would have any significant effecton potency. Indeed, compound 39 with 2-amino-5-(dimethylamino)gchloropyrazinoyl moiety at the P3 position resulted in a greater than 10-fold potency enhancement (ICs0 0.24 nM) compared to unsubstituted pyrazine derived compound 38. The removal of the 3-amino functionality from the pyrazine ring of compound 39 has significantpotency enhancing effect. As shown, compound 40 has displayed an enzyme inhibitory potency of 0.06 nM, a 4-fold improvementover 39. Further attachment of a basic amine functionality (cornpound 2) appears to have no effect on ICs0 value (0.07 nM) compared to compound 40. The marked enhancement of enzyme inhibitory potency of compounds 40 and 2 was translated into their antiviral potencies. Both compounds consistently exhibited an antiviral potency of 12 nM in cell culture assay. With the goal of minimizing peptide-like character while retaining comparable enzyme inhibitory and antiviral activity, we then explored the possibility of removal of the PrP3 amide carbonyl by attaching the 3’(R)-Thfg-derived amine directly to the pyrazine ring. This turned out to be a rewarding endeavor. Compound 27 thus obtained
,
I
Figure2. Stereoviewof theoptimizedboundconfomtionsofcompound 1(Ro31-8959)(pink)and the inhibitor23(green)superimposed in the L-689,502 inhibited HIV-1 protease active site.”
Potent HIV Protease Inhibitors
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16 2305
Table 111. Inhibitory Potencies (HIV-2) of Selected Compounds
amide carbonyl is removed. This has resulted in substantial improvement in antiviral potency while retaining the enzyme inhibitory potency at a level similar to that of compound 1. Further design of compounds from the study of ligand binding site interaction may lead to structurally novel inhibitors with less peptide-like character. Investigations along this line are in progress.
compd 1
2 23
ICW, nM (HIV-2)s 0.5 0.18 0.24
compd 26 27
ICW, nM (HIV-2)25 3.2 24.9
showed a similar level of enzyme inhibitory activity (ICs0 0.16 nM) as the inhibitor 1. More importantly, there is a dramatic improvement in antiviral potency (cIc95 3 nM)%of compound 27 compared to 1. Presumably, various functional groups present in the pyrazine ring of compound 27 are consequential to binding; however, the actual role of the pyrazine substituents has not been specifically examined. A thorough understanding of the contributions of the various functional groups in this binding region should await a detailed structure-activity relationship investigation. Intriguing changes in activity were observed upon introduction of 2-chromone carboxamide as the P3 ligand. Replacement of the 2-quinolinoyl group with a 2-chromone derivative (compound 41) resulted in 4-fold enhancement of both enzyme inhibitory and antiviral potencies compared to compound 26. Thus, simple replacement of Pz-asparagine and P3-2-quinolinoyl units with valine and 2-chromonoyl moieties afforded an inhibitor (compound 41) with comparable in vitro potencies to compound 1. Interestingly, substitution of valine in 41 with 3'(R)-Thfg has no effect on potency. Selected inhibitors from this present series have been evaluated for their ability to inhibit HIV-2 protease.26As shown in Table 111, inhibitor 23, derived from 3'(R)-Thfg (IC500.24 nM), is 2-fold more potent than compound 1 (IC500.5 nM). In contrast, replacement of asparagine in 1 with valine (compound 26) resulted in over 6-fold loss of potency. Furthermore, the N-pyrazinyl compound 27 showed an inhibitory potency of 24.9 nM. Compound 2 with 3'(R)-Thfg at the PZ and substituted pyrazinoyl moiety at P3 is the most potent inhibitor (HIV-2) in this series. This compound has exhibited an ICs0 value of 0.18 nM (HIV-2),nearly a 3-fold improvement over compound 1 (Ro 31-8959).
Conclusion Replacement of the Pz-asparagine and Pa-2-quinolinoyl ligand of present clinical candidate 1with 3'(R)-Thfg and substituted pyrazine derivatives has led to a novel series of potent inhibitors of HIV-1 and HIV-2 proteases. Interestingly, the inhibitor containing tetrahydrofuranylglycine with the BS,3R-configuration has exhibited nearly 100-fold potency enhancement over the 2S,3Sderived inhibitor. This marked difference in activity and strong stereochemical preference has suggested some specific hydrogen-bonding interaction with the residues in the Sz-binding domain of HIV-1 protease. Also, it has been demonstrated that substituted pyrazinoyl derivatives are effective as Pa-ligands, leading to potent protease inhibitors. Of particular interest, compound 2, with 3'(R)-Thfg at PZand pyrazine derivative a t P3, is one of the most potent inhibitors of HIV-1 and HIV-2 proteases. Since these are the genetically most divergent strains of HIV known to exist to date, the protease inhibitors which are potent against HIV-1 and HIV-2 strains may have a beneficial effect in terms of reducing susceptibility to clinical resistance. Another intriguing result in this series is the identification of compound 27 in which the Pz-P3
Experimental Section All melting points were recorded on a Thomas-Hoovercapillary melting point apparatus and are uncorrected. Proton magnetic resonance spectra were recorded on a Varian XL-300 spectrometer using tetramethylsilane as internal standard. Significant lH NMR data for representative compounds are tabulated in the following order: multiplicity (8, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), number of protons, coupling constant(s) in Hz. FAB mass spectra were recorded on a VG Model 7070 mass spectrometer, and relevant data are tabulated as m / z . Elemental analyses were performed by the analytical department, Merck Research Laboratories, West Point, PA, and were within &0.4% of the theoretical values. Anhydrous solvents were obtained as follows: methylene chloride, distillation from PlOlO; tetrahydrofuran, distillation from sodium/benzophenone; dimethylformamide and pyridine, distillation from CaH2. All other solventswere HPLC grade. The abbreviations DME, DMF', THF, HOBT and EDC refer to 1,2-dimethoxyethane, N,N-dimethylformamide, tetrahydrofuran, 1-hydroxybenzotriazole hydrate, and 1-ethyl-3-(3-(dimethylamino)propy1)carbodiimidehydrochloride. Column chromatography was performed with E. Merck 240-400 mesh silica gel under low pressure of 5-10 psi. Thinlayer chromatography (TLC)was carried out with E. Merck silica gel 60 F-254 plates. trans-4-Phenyl-2-buten-1-01 (4). A mixture of CuCN (2.43 g, 27 mmol) was added to a solution of butadiene monooxide (19 g, 270 mmol) in 500 mL of anhydrous tetrahydrofuran, and the mixture was cooled to -78 "C. Phenylmagnesium bromide solution in ether (32 mmol) was added dropwise to this mixture. The reaction mixture was warmed to 0 "C and was stirred until the reaction mixture became homogeneous. The reaction mixture was cooled to -78 OC, and 0.29 mol of phenylmagnesium bromide solution in ether was added dropwise over 30 min. The reaction mixture was allowed to warm to room temperature with stirring and then quenched by slow addition of saturated NHlCl(50 mL) followed by NHdOH (30 mL), saturated NH&l (200 mL), and HzO (100 mL). The aqueous layer was extracted with two 200mL portions of ethyl acetate. The combined organic layers were dried and concentrated. The residue was distilled under vacuum (0.1 Torr) at 100 "C to give trans-4-phenyl-2-buten-1-01 (4,38.9 9). 2(R),3(R)-Epoxy-4-phenylbuten-l-o1(5). A mixture of powdered 4-A molecular sieves (3 g), titanium tetraisopropoxide (1.5mL), anddiethylo-tartrate (1.1mL) inanhydrousmethylene chloride (350 mL) was cooled to -22 "C, and tert-butyl hydroperoxide solution in isooctane (210mmol) was added slowly with stirring. After 30 rnin at -22 "C a solution of 4 (15.3 g, 100mmol) in anhydrous methylene chloride (50 mL) was added dropwise for 20 min at -22 "C. The reaction mixture was aged at -22 "C in a freezer for 20 h. Water (40 mL) was added to the reaction mixture, and after 30 rnin at 0 "C, 30% NaOH in brine (6 mL) was added. The resulting mixture was stirred for 1h at room temperature. The organic phase was separated, and the aqueous layer was extracted with two 30-mL portions of methylene chloride. Combined organic layers were dried over Na&04, diluted with toluene (300 mL), and concentrated. Chromatographyonsilicagelwith40% ethylacetateinhexanegave(2R,3R)epoxy-4-phenylbutan-1-01 (5, 10.3 g). (2S,35)-2-Hydroxy-3-azid0-4-phenylbutan-l-o1(6). A solution of titanium tetraisopropoxide (5.6 mL) and azidotrimethylsilane (5.0 mL) in anhydrous benzene (100 mL) was refluxed for 5 h. To this refluxing mixture was added a solution of 2.6 in anhydrous benzene g of 2(R),3(R)-epoxy-4-phenylbutan-l-01(5) (10 mL). The reaction mixture was refluxed for 15 min, cooled
2306 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16 to room temperature, and quenched by addition of 5 % (150 mL). After the resulting biphasic mixture was stirred for 1 h, the organic layer was separated and the aqueous layer was extracted with two 20-mL portions of ethyl acetate. Combined organic layers were washed with saturated NaHC03 (50 mL), dried over MgS04,and concentrated to give the azido diol 6 (2.7 g) as a white solid mp 80-82 "C (lit! mp 80.5-82 "C). 3(S)-Azido-1,2(R)-epoxy-4-phenylbutane(7). The azido diol 6 (2.7 g, 13 mmol) was dissolved in chloroform (30 mL), and 2-acetoxyisobutyryl chloride (2.5 mL) was added. After the solution was stirred for 5 hat room temperature, saturatedsodium bicarbonate (50 mL) was added, and the resulting biphasic mixture was stirred for 10 min. The aqueous layer was extracted with two 30-mLportions of chloroform. Combined organic layers were dried over NazSO4 and concentrated, The residue was dissolved in anhydrous THF (10 mL), and solid NaOMe (1 g) was added. After the mixture was stirred for 3 h at room temperature, saturated NKCl(20 mL) was added and the mixture extracted with two 20-mL portions of ethyl acetate. Combined organic layers were dried over MgS04and concentrated. Chromatography on silica gel with 8% ethyl acetate in hexanes gave 3(S)-azido-l,2(R)-epoxy-4-phenylbutane(7, 1.75 g) as an oil: [@D = +14" (c = 1.5, MeOH); lH NMR (300 MHz) CDC13 6 7.4-7.2 (m, 5H), 3.6 (m, lH), 3.1 (m, lH), 2.95 (dd, lH, J = 4.6, 13.9 Hz), 2.8 (m, 3H). Diethyl (3(R)-Tetrahydrofurany1)malonate (9). To a stirred solution of 13 g (130 mmol) of 3(R)-hydroxytetrahydrofuran in 200 mL of methylene chloride cooled to -10 "C was added 31 mL of triethylamine and 13 mL of methanesulfonyl chloride, keeping the temperature below 5 "C. After 20 min at 0 "C,50 mL of water was added and stirring continued for 10 min. The mixture was diluted with 500mL of methylene chloride, and the layers were separated. The organic layer was washed with 20 mL of 6 N HC1, and the combined aqueous layers were extracted with two 50-mL portions of methylene chloride. The combined organic extracts were washed with 25 mL of saturated sodium bicarbonate, dried (MgSOd), and concentrated. After the extracts were dried further under vacuum (1mm) at room temperature for 30 min, 32.2 g of the mesylate was obtained. The mesylate was added to a solution of 5 molar equiv of diethyl sodiomalonate in dimethylformamide prepared by adding 110 mL of diethylmalonateto an ice-cold suspension of 29 g of sodium hydride 60% oil dispersion (washed with three 200-mL portions of hexanes) in 300 mL of dimethylformamide over 1 h. The resulting mixture was heated to 95 f 5 OC for 16 h and then cooled and quenched by addition of 120 mL of glacial acetic acid. The mixture was diluted with 1 L of ether and 2 L of water and separated. The aqueous layer was extracted with two 200-mL portions of ether. Combined ether extracts were washed with 50 mL of saturated sodium bicarbonate, dried (MgSOd), and concentrated. After removal of excess diethyl malonate by distillation through a 20-cm Vigreux column at 5-10 mm (bp 85 OC), the product was distilled through a short path at 1.5 mm, bp 115-20 "C. There was obtained 27.7 g of product as a colorless l i q ~ i d : [ a=] ~-21.2" ~ ~ (c = 1.0, MeOH). 'H-NMR (CDC13) 6 4.2 (m, 4H) 4.0 (dd, lH, J = 7, 8 Hz), 3.88 (m, lH), 3.75 (dd, lH, J = 7,15 Hz), 3.5 (dd, lH, J = 7,8 Hz), 3.3 (d, lH, J = 8 Hz), 2.9 6H). Anal. (C11HlsOd (m, lH),2.1 (m, lH), 1.68 (m, lH), 1.3 (m, C, H, N. Diethyl (3(S)-Tetrahydrofurany1)malonate. From 17 g (170 mmol) of 3(S)-hydroxytetrahydrofuranthere was obtained 33 g of product as a colorless liquid: [ ( u ] 2 3 ~ = +21.4" (c = 1.0, MeOH). Anal. (C11HlsOa) C, H, N. (3(R)-Tetrahydrofurany1)aceticAcid (10). A mixture of 27.7 g (120 mmol) of diethyl (3(R)-tetrahydrofuranyl)malonate, 400 mL of 1 N sodium hydroxide, and 200 mL of ethanol was stirred at room temperature for 48 h. After the ethanol was removed by concentration under reduced pressure, the aqueous solution was acidified to pH 1 with 6 N HC1 and concentrated to dryness. The solid residue was extracted with 500 mL of ethyl acetate, filtered, and concentrated to dryness. The resulting thick oil (diacid) and 0.9 g of CuzO were dissolved in 250 mL of anhydrous acetonitrile and heated to 80 "C for 6 h. The mixture was concentrated to dryness and the residue stirred with 40 mL of 6 N HC1 for 5 min and then extracted with three 250-mL portions of ethyl acetate. The organic extracts were dried
Ghosh et al. (MgSO4) and concentrated and the crude product (17 g) purified by evaporative distillation at 1mm (oven temperature 120-150 "C). There was obtained 13.6 g of product as a colorless liquid: [(Y]=D = -25" (c = 1.40, MeOH); 'H-NMR (CDCls) 6 3.98 (dd, lH, J=7,8Hz),3.88(m,lH),3.8(m,2H),3.45(dd,lH,J=7,8Hz), 2.62 (m, lH), 2.45 (d, 2H, J = 8 Hz), 2.15 (m, lH), 1.6 (m, 1H). Anal. (C6H1003)C, H, N. (3(S)-Tetrahydrofuranyl)acetic Acid. From 30 g (130 mmol) of diethyl (3(S)-tetrahydrofuranyl)malonatewas obtained 16.85 g of product as a colorless liquid [(U]=D = +25" (c = 1.44, MeOH). Anal. (C6HloOs) C, H, N. 3-(2-(3(R)-Tetrahydrofuranyl)-l-oxoethyl)-4( @-(phenylmethyl)-2-oxazolidinone(11). To a stirred solution of 2.6 g (20 mmol) of optically pure (3(R)-tetrahydrofuranyl)aceticacid in 20 mL of dry tetrahydrofuran was added 3.6 mL of triethylamine and then 2.7 mL of trimethylacetyl chloride. After 15 min at -15 "C, the reaction slurry was warmed to 0 "C over 20 min and then recooled to -78 "C. In a separate flask, 6.36 g of 4(S)-(phenylmethyl)-2-oxazolidinonewas dissolved in 100 mL of dry tetrahydrofuran and cooled to -60 "C. To this solution was added 23 mL of 1.6 M n-butyllithium in hexanes over 15 min. This cold solution was taken up in a syringe and added to the white slurry prepared as described above. After the mixture was stirred for 1h at -78 "C, 20 mL of 1N sodium bisulfate was added. The tetrahydrofuran was removed under reduced pressure, and the remaining aqueous mixture was extracted three times with 100-mLportions of methylene chloride. The combined extracts were washed with 20 mL of saturated sodium bicarbonate, dried (MgSOr), and concentrated to dryness. Purification by chromatography on silica gel with 35% ethyl acetate in hexanes gave 5.4 g of product as a thick oil. Anal. (CleHlsNO4) C, H, N. 34243(5)-Tetrahydrofurany1)-1-oxoethy1)-4(S)-(phenylmethyl)-2-oxazolidinone. From 2.6 g (20 mmol) of optically pure 3(S)-tetrahydrofuranylacetic acid was obtained 5.4 g of product as a white crystalline solid: mp 76-77 "C. Anal. (CisHlsNoh) C, H, N. 3-(2(S)-Azido-2-(3(R)-tetrahydrofuranyl)-l-oxoethyl)-4(S)-(phenylmethyl)-2-oxazolidinone(12). A cooled (-78 "C) solution of 5.4 g of stereochemically pure 11 in 66 mL of dry tetrahydrofuran was added to a cooled (-78 OC) solution of 30 mL of 0.69 M potassium bis(trimethylsily1)amide in toluene diluted with 66 mL of dry tetrahydrofuran, keeping the temperature below -60 "C. After 30 min at -78 "C,a precooled solution of 7.34 g of 2,4,6-triisopropylbenzenesulfonylazide in 45 mL of dry THF was added. After 2 rnin at -75 "C, 5.4 mL of glacialaceticacid was added and the mixture warmed immediately to 30 "C for 1h. The solution was partitioned between 200 mL of methylene chloride and 100 mL of dilute brine. The aqueous phase was extracted with three 75-mL portions of methylene chloride. Combined organic extracts were washed with 100 mL of saturated sodium bicarbonate, dried (MgSOd), and concentrated. Chromatography on silica gel with 1:9 ethyl acetatemethylene chloride gave 5.3 g of product as a thick oil. 3-(2(S)-Azido-2-(3( S)-tetrahydrofurany1)-l-oxoethyl)-4(S)-(phenylmethyl)-2-oxazolidinone: mp 108-109.5 "C (softens at 95 "C). 2(S)-Azido-2-(3(R)-tetrahydrofuranyl)acetic Acid (13). To a stirred solution of 5.2 g of 12 in 110 mL of THF was added 37 mL of water and 0.78 g of LiOH. After 45 min at room temperature 2.77 g of NaHC03 was added and the mixture concentrated to remove THF. The aqueous solution was diluted to 80 mL and extracted with two 50-mL portions of methylene chloride. The aqueous layer was made acidic with 5 mL of concd HC1 and extracted with four 50-mL portions of ethyl acetate. Combined ethyl acetate extracts were dried (MgSO4) and concentrated. After drying there was obtained 2.9 g of product as a white crystalline solid. Sublimation at 0.1 mm, 80-90 "C, gave 2.78 g of pure 13: mp 83-5 "C; [(r]=D = -92.9" (c = 1.75, MeOH); 'H-NMR (CDC13)6 9.5 (br s, lH), 3.8 (m, 3H), 2.75 (m, lH, J = 8 Hz), 2.15 (m, lH), 1.85 (m, 1H). Anal. (C~HsN303) C, H, N. (ZS,3'R)-3'-Tetrahydrofuranylglycine (13a). A solution of 400 mg of (2S,3'R)-2-azido-2-(3'-tetrahydrofuranyl)aceticacid in 20 mL of ethanol and 10 mL of H20 was stirred under an atmosphere of hydrogen over 50 mg of 5 % Pd/C for 24 h. Removal of the catalyst by filtration and concentration under reduced
Potent HZV Protease Inhibitors
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16 2307
pregeure gave, after drying, 355 mg of product as a white solid ture of 0.050 g of 2(S)-azido-(3(R)-tetrahydrofuranyl)aceMc acid 14% = -17.7' (C 1.29, H2O); 'H-NMR (CDQ,) 6 3.7-4 (m, (13), 0.10 g of the amine 20,0.034 g of 1-hydroxybenzotriazole 5H), 2.75 (m, lH), 2.15 (m, lH), 1.9 (m, 1H). Anal. (Callhydrate, and 5 mL of dmethylformamide was added 0.055 g of NOa-0.2EtOH) C, H, N. l-ethyG3-(3-(dimethylamino)propyl)carbodiimidehydrochloride and 0.027 mL of 4-methylmorpholine. After being stirred for 4 2(S)-Azido-2-(3(S)-tetrahydrofuranyl)acetic Acid. The h, the mixture was concentrated to dryness under reduced product was a thick oil: [a]?$ = -107O (c = 1.75, MeOH); 'Hpressure. The residue was partitioned between 50 mL of ethyl NMR (CDCls) S 8.3 (br 8, lH), 3.9 (m, 3H), 3.8 (dd, 1 H , J = 8, acetate and 20 mL of water. The organic layer was washed with 15 Hz), 3.72 (dd, lH, J = 7,8 Hz), 2.75 (m, lH, J = SHz), 2.15 20 mL of saturated NaHCOs, and the combined aqueous layers (m, lH), 1.92 (m, 1H). Anal. (CsHgNaOs) C, H, N. were extracted with 20 mL of ethyl acetate. Combined organic (2S,3'S)-3'-Tetrahydrofuranylglycine. A solution of 300 extractswere dried (MgSO3and Concentrated. Chromatography mg of (2S,3'S)-2-azido-2-(3'-tetrahydrofuranyl)aceticacid in 20 on silica gel eluting with ethyl acetate gave 0.150 g of azido amide mL of ethanol and 10mL of HsO was stirred under an atmosphere 21 as a thick oik 'H-NMR (CDCb) 6 7.1-7.4 (m, 5H), 6.7 (d, lH, of hydrogen over 50 mg of 5% Pd/C for 24 h. Removal of the J = 8.8 Hz),5.8 (bra, lH), 4.35 (m, lH), 3.9 (m, lH), 3.8 (brd, 2H, catalyst by filtration and concentration under reduced pressure J = 5.6 Hz),3.5-3.7 (m, 2H), 3.34 (dd, lH, J = 6.6, 6.1 Hz), gave, after drying, 270 mg of product as a white solid [all$ = 2.9-3.05 (m, 4H), 2.5-2.7 (m, 2H), 2.5-2.25 (m, 3H), 1.2-2.1 (m, +10.3O (c = 1.125, H2O); 'H-NMR (CDCL) 6 3.6-4 (m, 5H), 2.72 (m, lH), 2.2 (m, lH), 1.85 (m, 1H). Anal. (C&I~NOS.O.~E~OH) 15H), 1.35 (e, 9H). C, H, N. The azido amide 21 was dissolved in 20 mL of THF and 5 mL Methyl 3-Bmmo-S-(4methylpipera~yl)-6chloro-2-pyraofmethanol,and5Omgof5% palladiumoncarbon wassuspended in the mixture. The resulting mixture was stirred under an zinecarboxylate (16). To a stirred suspension of 11.3g of methyl atmaaphere of hydrogen for 12 h. Removal of the catalyst by 3-amino-6.(4-methylpiperazinyl)-6-chloro-2-pyrazinoate~2 (1Sb) fiitration and the solvents by concentration under reduced in 75 mL of 48% HBr and 120 mL of glacial AcOH cooled in an pressure gave the amine 22 (0.13 g). ice bath to 5 "C was added a solution of 6 mL of bromine in 10 mL of AcOH over 45 min with efficient stirring. To this mixture N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-3(~-( (N-(2was added a solution of 8.3 g of sodium nitrite in 18mL of water, quinolylcarbonyl)-2( S)-(3(~tetrahyclrofu~yl)g~cinyl)maintaining the temperature below 8 "C. After addition was amiao(butyl)decahydro-(~S,8aS),isoquinoline-3(S)-carcomplete the mixture was stirred for 30 min, and then excess boxamide (23). A solution of 0.056 g of diphenylphoaphinic bromine was destroyed by dropwise addition of 100 mL of 30 % chloride in 2 mL of dry THF was added to a precooled (-10 O C ) NaHSOa. The solution was neutralized to pH 8 by dropwise solution of quinaldic acid (60 mg) and triethylamine (0.042 mL) addition of 20% NaOH (ca. 200 mL) and then extracted with 5 in 10 mL of dry THF. After the mixture was stirred for 1h at X 100 mL of CH2Cl2. The organic extrade were washed with 50 -10 OC, a solution of 0.130 g of the amine 22 in 5 mL of THF was mL of dilute N h O H , dried over MgSO4, and concentrated to added and the mixture allowed to warm to room temperature dryness. After drying under vacuum, there was obtained 6.3 g and stir for 3 h. The solvents were removed under reduced (47%) of the bromide as a yellow crystallinesolid mp 113-4 OC. pressure and the residue partitioned between 50 mL of ethyl acetate and 10 mL of dilute NaHCOs. The organic layer was Anal. (C11H1&cw402) C, H, N. 8-(4-Methylpiperaainyl)-6-chloro-2-pyrazinecarboxylic dried (Mgso4) and concentrated under reduced preasure. ChroAcid Hydrochloride (17). A solutionof 2.6g of methyl 3-bromomatography on silicagel elutingwith4 % methanol in ethyl acetate gave after trituration with ether-hexanes 0.160 g of product as 5-(4-methylpiperazinyl)-6-chloro-2-pyrazinecarboxylate (16) in a white amorphoussolid. A sample c r y s t d b d from ethyl acetate 100 mL of THF was stirred under a balloon filled with hydrogen as colorlege prisms (hemihydrate): mp 2 0 6 - 6 O C ; 'H-NMR over 0.26 g of 5% palladium on carbon for 2 days. The catalyst (CDCls) S 8.7 (d, lH, J 8.3 Hz),8.35 (d, lH, J 8.4 Hz),8.25 was removed by filtration, and the residue after concentration (d,lH,J=8.4Hz),8.15(d,lH,J=8.4Hz),7.9(d,lH,J=8.5 was partitioned between saturated NaHCOs and ethyl acetate. Hz), 7.8 (t, lH, J = 6.8 Hz), 7.65 (t,lH, J = 6.8 Hz),6.96-7.1 (m, The organic extracts were dried over MgSO4 and concentrated. 4H), 6.9 (t, lH, J = 7.6 Hz), 6.7 (br d, lH, J = 8.8 Hz), 5.97 (brs, Chromatography of the residue on silica gel eluting with 5% 1H),4.5 (dd,lH,J= 7.2,7.8Hz),4.3 (m,lH),3.9 (m,2H),3.&3.7 MeOH in CH2Cl2 gave 1.42 g (70%) of a yellow solid mp 96-7 (m, 4H), 2.6-3.1 (m, 4H), 2.3 (m, 2H), 1.7-2.1 (m, 6H), 1.2-1.6 "C. Anal. (C&&1N&) C, H, N. A solution of 0.5 g of methyl 5-(4-rnethylpiperazinyl)-6-chloro(m, 9 H), 1.3 (8,gH). Anal. (C&&505*0.5H20) C, H, N. N-tert-Butyl-2-[2(R)-hydmxy-4-pheny1-3(S)-( (2(S)-ami2-pyrazinecarboxylateand 6 mL of 1N NaOH in 20 mL of EtOH no-2-(3(s)-tetr)~l)butyl)but was allowed to stir at room temperature overnight. The mixture was acidifiedto pH < 2 with 6 N HCl and concentratedto drynew. 8aS)-3(S)-carbo&de. Followingtheprocedurefor compound The solids were stirred with 200 mL of 10% EtOH in CHCh and 22, 0.050 g of 2(S)-azido-2-(3(S)-tetrahydrofuranyl)acetic acid was coupled to 0.10 g of the amine 20 to provide 0.132 g of the filtered, and the filtrate was concentrated to dryness. The offdiastereoisomer of 21 that contains the 2(s>-azido-3(s>-tetrahywhite solid product weighed 0.52 g (96 % ) and was homogeneous drofuranyl moiety: 'H-NMR (CDCb) S 7.15-7.4 (m, 5H), 6.7 (d, by HPLC. 1 H , J = 8.7 Hz),5.8 (brs,lH),4.3 (m,lH), 3.9 (m,lH),3.3-3.8 N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-S( S)-azidobuty1)(m, 5H), 2.9-3.02 (m, 3H), 2.6 (m, 2H), 2.45 (m, lH), 2.3 (m, 2H), decahydro(4aS,8aS)-isoquinoline-3(S)-carbo.amide (19). A 1.2-2.1 (m, 15H), 1.35 (8, 9H). mixture of 8.46 g of N-tert-butyldecahydro-(4aR,8aS)-isoquinoline-3(S)-carboxamide(18) ([a]% = -70 "C (c = 1.0, MeOH)) The above azido amide was hydrogenated over 6 % palladium and 10.3 g of 3(S)-azido-l,2(R)-epoxy-4-phenylbutane(7) in 200 on carbon to provide the titled amine (0.12 8). mL of 2-propanol was heated to 80 "C overnight and then N-tert-Butyl-2-(2( @-hydroxy-4-phenyl-3(8)-( (N-(2concentrated to dryness under reduced pressure. Recrystalliquinolylcarbonyl)-2(S)-(3( S)-tetrahydmfuranyl)glycinyl)zation from ethyl acetate-hexanes gave 9.63 g of product: mp amino)butyl)decahydro-( 4aS,8aS)-iroquinoline-S(@-car149-50OC; [ala$= -97.8O (c = 1.1,MeOH). Anal. (CuHs,N~02) boxamide (24). A sample crystallized from ethyl acetate C, H, N. (hemihydrate): mp 206-6 "C; 'H-NMR (CDCb) S 8.5 (d, lH, J N-tert-But y ldecahy dro-2- (2(It)-hydroxy-4-phenyl-3( S)= 8.8 Hz), 8.35 (d, lH, J = 8.3 Hz), 8.25 (d, lH, J = 8.4 Hz),8.1 aminobutyl) (4aS,8aS)-ieoquinoline-3( S)-carboxamide (20). fd, lH, J = 8.4 Hz), 7.9 (d, lH, J = 8.5 Hz), 7.8 (t, lH, J = 7.2 A solution of 5.0 g of 19 in THF (200 mL), MeOH (50 mL), and Hz), 7.65 (t, lH, J = 7.2 Hz),6.85-7.1 (m, 4H), 6.7 (m, 2H), 6.0 acetic acid (2 mL) was shaken with 1 g of 10% palladium on (brs, 18), 4.5 (t,lH, J = 8.8 Hz),4.25 (m, lH), 3.5-3.9 (m, 5H), carbon catalyst under an atmosphere of hydrogen for 12 h. 2.6-3.1 (m, 4H), 2.3 (m, 2H), 1.3-1.9 (m,16H), 1.3 (s,9H). Anal. Removal of the catalyst by fiitration and concentration under (CuJ&"Os.O.5H20) C, H, N. reduced pressure gave 4.68 g of product as a white solid mp N tert-B ut yl-2-(2(R)hydroxy-4-phenyl-3( S)-((N((6-(4165-166OC;[all$= -108O (c = 1.0,MeOH). Anal. ( C d & & ) met hylpiperazinyl)-6chloro-2-pyrasinyl)carbyl)-2(S)C, H, N. (3(R)-tetrahyclrofuranyl)glycinyl )amino)buty1)deoohydroN-ter&Butyl-2-[ 2(R)-hydroxy-4-phenyl-3( S)-((2(@-ami(4aS,8aS)-iroquinoline-3(S)-car~xamide (2). A mixture of no-z(3(R)-tet~~iuranyl)acetyl)butyl)d~~(~,-80 mg of 5-(4-methylpiperazinyl)-6-chloro-2-pyrazinecarboxylic 8aS)-isoquinoline-3( S)-carboxamide (22). To a stirred mixacid hydrochloride (17), 52 WLof diphenylphosphinic chloride,
2308 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16
C h 8 h et al.
and 80 NL of EtgN in 5 mL of THF was stirred at -10 OC for 2 After being stirred 12 h at 23 OC, the mixture was concentratad h. To this mixture was added 120 mg of amine 22 in 5 mL of to dryness under reduced pressure. The residue was partitioned THF. After being stirred overnight at room temperature, the between 100mL of ethyl acetate and 40 mL of water. The organic mixture was diluted with 50 mL of ethyl acetate, washed with layer was washed with 30 mL of saturated NaHCOa, and the 10 mL of saturated NaHCOs and 10 mL of dilute aqueous "3, combined aqueous layers were extraded with 50 mL of ethyl and concentrated to dryness. Preparative thin-layer chromaacetate. Combined organic extracts were dried (MgSOd and tography on silica gel eluting with 15% MeOH in CHCb gave 80 concentrated. Chromatography on silica gel eluting with 50% mg of an off-white solid mp 115-8 "C; 'H-NMR (CDCb) 6 8.76 ethyl acetate in hexane provided 0.126 g of azido amide 29 (Rj = 0.2) and 0.105 g of azido amide 30 (Rj = 0.43) as a thick oil. (m, lH), 7.78 (m, lH), 7.1-7.3 (m, 5H), 6.7 (m, lH), 4.4 (m, lH), Compound 2 9 1H-NMR (CDCW 6 7.15-7.35 (m, 5H), 6.7 (brd, 4.3 (m, lH), 3.5-3.9 (m, llH), 2.7-2.9 (m, 12H), 2.5 (m, 3H), lH, J = 8.6 Hz), 5.8 (brs, lH), 4.35 (brs, lH), 3.9 (m, 2H), 2.9-3.1 1.3-2.0 (m, 14 H), 1.3 (e, 9H). Anal. (CloH&lNaOa) C, H, N. (m,4H), 2.4-2.7 (m,6H),2.2-2.4 (m, 5H), 1.4-1.95 (m, 12 H), 1.35 Ntart-Butyl-2-(2(R)-hydro~~p~~l-3(S)-((N-( (2meth(e, 9H). Compound 3 0 'H-NMR (CDCb) 6;7.2-7.4 (m, 5H), 6.7 oxy~bonyl)-3-amino6-chloro-&pyrazinyl)-2( S)-(Q(R)-tet(brd, lH, J = 8.5 Hz),5.85 (brs, lH), 4.3 (brs, lH), 3.9 (brs, lH), rahydrofurany1)glycinyl)amino) buty1)decahydro-(4aS,3.85 (brd, lH, J = 6.2 Hz),3.4 (brs, lH), 2.8-3.05 (m, 4H), 2.558aS)-isoquinoline-3(S)-carboxamide (27). A mixture of 0.10 2.8 (m, 5H), 2.5 (m, lH), 2.3 (m, 4H), 1.4-1.9 (m, 12H), 1.35 (8, g of N-tert-butyl-2-(2(R)-hydroxy-4-phenyl-3(S)-((2(S)-(3(R)9H). tetrahydrofurany1)glycinyl)amino) butyl)decahydro-(4&,8&)N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-3( S)-( (N-(2isoquinoline-3(S)-carboxamide and 0.10 g of methyl 2-aminoquinolylcarbonyl)-2(S)-(3(R)-tetrahydrothiofuranyl)glyci4,5-dichloropyrazinoatein 10 mL of 2-propanol was heated to reflux overnight. After removal of solvents, the residue was ny1)amino)butyl)decahydzo-(4aS,8aS)-isoquinollne-3(~carboxamide (31). The azide derivatives 29 and 30 described purified by preparative thin-layer chromatography(5% methanol in ethyl acetate). Trituration with ether gave 23 mg of an above were converted into 31 and 32 using the procedures amorphous solid mp 265 "C dec; 'H-NMR (CDCls) 6 8.5 (m, described above for compound 23. The homers were separated lH), 7.1-7.3 (m, 6H), 6.3 (m, lH), 5.8 (m, lH), 5.7 (m, lH), 4.5 by silica gel chromatography (90%ethyl acetate in hexane), and (m, lH), 4.3 (m, 2H), 3.8-3.9 (m, 5H), 3.5-3.7 (m, 5H), 2.5-3.1 the more polar isomer (Rf0.72) was obtained as a crystalline solid (hemihydrate): mp 129-31 OC; 'H-NMR (CDCW d 8.7 (d, (m, 4H), 2.G2.2 (m, 2H), 1.2-1.9 (m, 14H), 1.3 (8, 9H). Anal. lH, J = 8.1 Hz),8.35 (d, lH, J = 8.3 Hz), 8.25 (d, lH, J = 8.3 (C&pClN&&2CHC&) C, H, N. Hz), 8.15 (d, lH, J = 8.5 Hz),7.9 (d, lH, J = 8.6 Hz), 7.82 (t, lH, 2( S)-Azido-2-(3(SR)-tetrahydrofuranyl)aceticAcid (28). J = 6.6 Hz), 7.67 (t, lH, J = 6.8 Hz), 6.95-7.2 (m, 4H), 6.8 (br A suspension of NaH was prepared by washing 60% NaH (1.9 g, 48 mmol) with hexane (2 X 10 mL), and toluene (40 mL) was d, lH, J = 8.7 Hz), 6.0 (brs, lH), 4.6 (t, lH, J = 7.5 Hz), 4.3 (m, lH), 3.9 (m, lH), 2.45-3.1 (m, 6H), 2.1-2.4 (m, 4H), 1.7-2.1 (m, added to the residue. To this was added triethyl phosphonoac7H), 1.2-1.6 (m, 10 HI, 1.3 (8, 9H). Anal. (CloH~Ns0,etate (8.7 g, 39 mmol) dropwise at 24 OC via an addition funnel. S.0.15CHCb) C, H, N. The reaulting mixture was stirred for 30 min and then cooled to 20 OC, and a solutionof thiofuran-3-one (4.0 g, 39 mmol) in toluene N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-3(8)-( (N-(2quinolylcarbonyl)-2(S)-(3( S)-tetrahydrothioiyl)g~ci(15 mL) was added dropwise, keeping the internal temperature between 24 and 26 OC throughout the addition. The mixture nyl)amino)butyl)decahydro-(4aS,8aS)-i~oquinoline-3(S)was allowed to stand for 10 min after the complete addition and carboxamide (32). The less polar isomer (Rj 0.81) was then quenched with water (100 mL), and the layers were crystallized from ethyl acetate (hemihydrate): mp 108-11 "C; 'H-NMR (CDCb) 6 8.5 (d, lH, J = 8.7 Hz), 8.35 (d, lH, J = 8.4 separated. The aqueous layer was extracted with ether, and the combined organic layers were dried over anhydrous Na804. Hz),8.25(d, lH, J = 8.4Hz),8.15(d, lH, J = 8.3Hz),7.9(d,lH, J = 8.3 Hz), 7.8 (m, lH), 7.65 (t, lH, J = 7.4 Hz), 6.95-7.15 (m, Filtration and concentration under reduced pressure afforded crude product which was chromatographedover silica gel to afford 4H),6.7(m,2H,J=7.0Hz),6.0(brs,lH),4.6(t,lH,J~8.6Hz), 4.3 (m, lH), 3.9 (m, lH), 2.5-3.1 (m, 6H), 2.3 (m, 2H), 1.3-2.1 (m, the unsaturated ester (5 g) as an oil. 18H), 1.3 (8, 9H). A d . (C&&0&0.45EtOA~) C, H, N. To a solution of the unsaturated ester (2.5 g, 14.5 mmol) in 3:l ethyl acetate/methanol (100 ml) was added 10% palladium on N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-3(S)-((N-(2quinolylcarbonyl)d( S)-(3(R)-diosotetrahydrothioturanyl)charcoal (0.6 g), and the resulting mixture was hydrogenated at 50 psi for 12 h. After this period, the catalyst was fiitered off, glyciny1)amino)butyl)decahydro-(4aS,8aS)-isoqdnolineand the filtrate was concentrated under reduced pressure. The 3(S))-carboxamide(33). To a stirred solution of compound 31 resulting reeidue was chromatographed over silica gel to provide (32.4 mg) and 4-methylmorpholiie N-oxide (16.2 mg) in acetone the saturated ester (0.66 g) and recovered unsaturated ester (1.5 (2 mL) and water (1mL) at 0 "C was added a solution of osmium tetraoxide (2.5%)in 2-methyld-propanol(O.1mL). Theresulting I?). To a solution of saturated ester (0.66 g, 3.8 mmol) in DME (10 mixture was stirred at 24 OC for 24 h and then Concentrated mL) was added 1M LiOH (10 mL), and the resulting mixture under reduced pressure. The residue was partitioned between was stirred for 12 h at 24 OC. The mixture was then acidified ethyl acetate (50 mL) and water (20 mL), and the layers were with concd HCl and extracted with ethyl acetate (2 X 50 mL). separated. The organic layer was washed with brine and dried The combined organic extracts were dried over Na804. Filover anhydrous NaaOb Filtration and evaporation of the solvent under reduced pressure gave a residue which was purified by tration and concentration provided the (3(SR)-Tetrahydrothiosilicagel chromatography (90% ethyl acetate in hexane) to afford furany1)acetic acid (0.6 g): lH NMR (CDCla) 6 3.0-3.1 (m, lH), 2.9(m,2H),2.5-2.6(m,2H),2.5(m,2H),2.2(m,1H),1.7(m,1H). 19 mg of compound 33 as a white solid: mp 133-34 OC. Anal. (C&N50&0.75MeOH) C, H, N. From 0.6 g of (3(SR)-tetrahydrothiofuranyl)acetic acid was obtained 0.54 g of the title azido acid 28 following asymmetric N-tert-Butyl-2-(2(R)-hydrory-4-phenyl-S(S)-( (N-(2azidation and hydrolysis procedures as mentioned above (comquinolylcarbonyl)-2(S)-(3(s)-dioxotetrahydrothiofuranyl)glyciny1)amino)butyl)decahydro-( 4aS,8aS)-ieoquinolinepounds 12 and 13): 'H NMR (CDCb + CDsOD) 6 3.9 (m, lH), 3.65 (m, 1H),2.9 (m,2H),2.8 (m, lH), 2.6 (m, lH), 2.2 (m, lH), 3(S)-carbodde (34). Following the above oxidation pro1.9 (m, 1H). cedure, 16.4 mg of compound 32 was converted to sulfone derivative34 (10.4 mg). The product was obtainedas a crystalline N-tert-Buty1-2-(2(R)-hd~~-~p~nyl-3(5)-( (2(S)-azidosolid (hemihydrate): mp 128-30 OC. Anal. (Cds$JsOe2-(3(R)-tetrahydrothiofuranyl)acetyl)amino)butyl)decahydro-(4a5,8aS)-iroquinoline-3(S)-~rbo~mide (29) S.O.lSCHCb, 0.5 MeOH) C, H, N. and N-tert-Butyl-2-(2(R)-hydroxy-4-phemyl-3(~-((2(S)-azi4-TetrahydropyranylaceticAcid. To a stirred, cooled do-2-(3(S)-tetrahydrothiofuranyl)acetyl)amino)butyl)suspension of 0.9 g of sodium hydride (60% oil dispersion) in 5 decahydro-(4aS,8aS)-ieoquinoline-3(S)-carboxamide (30). mL of benzene was added 5.32 g of triethyl phosphonoacetate in To a stirred mixture of 0.150 g of 2(R)-azido-2-(3(SR)-tetrahy3 mL of benzene, keeping the temperature below 30 "C. After drothiofurany1)aceticacid (28),0.270 g of the amine 20,0.135 g 1h at 25 OC, a solution of 2 g of tetrahydro-4H-pyran-4-onein of 1-hydroxybenzotriazolehydrate, and 5 mL of dimethylfor3 mL of benzene was added slowly. After 1h at 25 OC, 20 mL mamide was added 0.191 g of l-ethyl-3-(3-(dimethylamino)of water and 50 mL of ether were added. The organic layer was propy1)carbodiimidehydrochloride and 0.5 mLof triethylamine. dried over MgSO, and concentrated to dryness. Evaporative
Potent HIV Protease Inhibitors distillation at 5 mm, oven temperature 130 OC, gave 1.8 g of product as a fragrant liquid. Hydrogenation in 20 mL of ethanol with 0.5 g of 10% palladium on carbon under 1atm of hydrogen for 10 days gave 1.8 g of saturated product after fiitration and concentration. Saponification using 20 mL of ethanol and 10 mL of 10% NaOH for 4 h followed by acidification (H~SOI)and ether extraction gave 1.5 g of product. Evaporative distillation at 0.1 mm, oven temperature 160-8 "C, gave 1.1g of product as a crystalline solid mp 61-2 "C. Anal. (C7HllOa) C, H, N. N-tert-Butyl-2-(2(S)-hydroxy-4-phenyl-3(S)-( (N-(2quinolylcarbonyl)-2(@-(4-tetrahydropyranyl)glycinyl)-
Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16 2309
containing medium (1OOO mg/mL, Amgen). The lymphocytes were activated with phytohemaglutinin (5 mg/mL, Sigma) prior to virus infection and were maintained in medium containing IL-2 (lo00 mg/mL, Dupont). The measurement of viral expreesion was performed by fixed cell immunofluorescence usingantiHIV-1 human serum. Measurement of Viral Spread. The measurement of viral expressionwas performed by either fiied cell immunofluoreecence using anti-HIV-1 human serum or p24 ELISA. In the immunofluorescenceaway the loweat concentration of compoundwhich completely prevented the spread of the virue from initially amino)butyl)decahydro-(4aS,8a@-~oquinoline-3(@-carinfected cells at 7-24 days poet infection was defined as the CIC boxamide (35). The 4-tetrahydropyranylaceticacid described (cell culture minimal inhibitory concentration). above was converted into the 2(S)-azido-2-(tetrahydropyranyl)In the p24 ELISA assay, the cell culture inhibitory concenacetic acid and 35 using the procedures described above for tration (CIC) is defiied as the concentration which inhibited by compounds 13and 23. The product was obtained as a crystalline greater than 95 % the spread of infection, as aeseeeed by agreater solid (hemihydrate): mp 122-4"C. Anal. (C~IH~~NF,OF,-O.~~HZO) than 95% reduction in p24 antigen production relative to C, H, N. untreated controls. Using a multichannel pipettor, the settled N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-3(S)-( (N-(2cells were resuspended, and a 125-mL aliquot was harvested into quinolylcarbonyl)-2( S)-(3(R)-tetrahydrofuranyl)glycinyl)a separate microtiter plate. After the settling of the cells, the amino)buty1)decahydro-( 4aS,8aS)-isoquinolhe-3( @-carplates were frozen subsequent to m a y of the supernatant for boxamide (37). From quinoxaline-2-carboxylicacid and 22, using HIV p24 antigen. The concentration of p24 antigen was measured the procedures described above for 2, was obtained a white solid by an enzyme immunoassay, described as follows. Aliquota of C, H, N. mp 207-8 OC. Anal. (C~~H~ZN~OC,-O.~E~OAC) p24 antigen to be measured were added to microwells coated N-tert-B~tyl-2-(2(R)-hy~~~-pheayl-3( S)-((N-( tpmziwith a monoclonal antibody specificfor HIV core antigen. The nylcarbonyl)-2(@-(3(R)-tetrahydrofuranyl)glycinyl)amimicrowells were washed at this point and at other appropriate no)butyl)decahydro-( 4aS,8aS)-isoquinoline-3(@-carboxasteps that follow. Biotinylatsd HIV-specific antibody was then mide (38). From pyrazine-2-carboxylic acid and 22, using the added, followed by conjugated streptavirudi-horseradish perprocedures described above for 2, there was obtained a white oxidase. A color reaction occurs from the added hydrogen solid mp 105-9 "c. Anal. (C&&O6*0.6CHC&) c, H, N. peroxide and tetramethylbenzidine Substrate. Color intensity ie N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-3(S)-( (N-((&(dim- proportional to the concentration of HIV p24 antigen. ethylamino)-6-chloro-2-pyrazinyl)carbonyl)-2(@-(3(R)-tetMolecular Modeling. All modeled structurea we& built using rahydrofuranyl)glycinyl)amino)butyl)decahydro-( 4aS,the Merck molecular modeling program AMF (Advanced Mod8a@-isoquinoline-3(@-carboxamide (39). From methyl elingFacility)" and energy minimized usingthe Merck molecular 3 - b r o m o - b ( d i m e ~ y ~ o ) - 6 ~ 0 r o - 2 - p ~ ~ and b o ~ l a tforce e ~ ~field, OPTIMOL, which is a variant of the MM2 program. 22, using the procedures described above for 17 and 2, was all Due to the low pH optimum of the HIV-1 protease,obtained a white solid mp 101-3 "C. Anal. (Cs7HsClNr~Ovtitratable residues were charged in the calculations with the O.5EtOH) C, H, N. exception of Tyr59 and one of the pair of catalytic aspartic acids, N-tert-Butyl-2-(2(R)-hydroxy-4-phenyl-3(@-( (N-( (&(dimA s ~ 2 5 .During ~ ~ energy minimizations the enzyme-active site ethylamino)-6-chloro-2-pyrazinyl)carbonyl)-2(S)-(t(R)-tetwas held fiied at the X-ray geometry. rahydrofuranyl)glycinyl)amino)butyl)decahydro-( 4aS,Graphics visualizationand molecular surface calculations were 8aS)-isoquinoline-3(@-carboxamide (40). From methyl performed usingQuanta.82 All molecular surfaces were generated 3 - a m i n o - 5 - ( ~ e ~ y ~ 0 ) - 6 ~ ~ 0 ~ 2 - pand y r ~ iusing n ~ a~ neutral ~ ~ t e0.95-A ~ radius probe with contouring performed 22, using the procedures described above for 17 and 2, was at an interaction energy of 0 kcal/mol. obtained a white solid (60%) after recrystallization from ethanol-water: mp 115-7 "c. Anal. (C~H&lN~06.2.0-AcOH) Acknowledgment. The authors gratefully acknowC, H, N. ledge the assistance of Dr.H. Ramjit for mass spectra, J. Biology. Inhibition of HIV-1 Protease. The ICINvalues P. Moreau for elemental analysis, and Ms. Jean Kaysen were determined using purified HIV-1 protease.21 Inhibition of the cleavage of the peptide H-Val-Ser-Gln-Am-(L-b-naphthy- for help in manuscript preparation. lalanine)-Pro-Ile-Val-OHwas assessed at 30 "C, pH = 5.5 with Referenoes [Enz] = 30 pM and BSA along with the inhibitor. The reaction was quenched with HpO,, and the products were analyzed by (1) Kohl,N. E.;Emiii, E. A.; Schleif,W. A.; Dad, L. J.; Heimbach, using HPLC with UV detection (225 nm)for quantification of J. C.; Dixon, R. A. F.; Scholnick, E. M.; Sigal, I. S. Active Human Immunodeficiency Virus Protease is Required for Vial Infectivity. the products. For ICaodetermination, a substrate concentration R o c . Natl. Acad. of Sci. U.S.A. 1988,86,4888-90. of 0.4 mg/mL was used, and the data were fit to a four-parameter ( 2 ) (a) Huff, J. R. HIV Protease: A Novel ChemotherapeuticTarget sigmoidal equation. The results of these measurements are shown for AIDS. J.Med. Chem. 1991,34,230614.(b) Norbeck, D.W.; in-Tables 1-and 11. Kempf, D. J. HIV Protease Inhibitors. In A n w l Reports in Inhibition of HIV-1 Viral Spread in Cell Culture. Selected Medicinal Chemistry; Bristol, J. A., Ed.; Academic Pres New York, 1991;Vol. 26,pp 141-150. (c) Tomawlli, A. G.; Howe, W. compounds were dissolved in dimethyl sulfoxide and serially J.; Sawyer,T.K.;Wlodawer,A.; Heinribon,R.L. The complexities diluted into cell culture medium to achieve the test concentraof AIDS An heesment of the HIV Proteaee as a Therapeutic tions. Cells were infected and grown in a medium of RPMI-1640 Target. Chimicaoggi1991,(May),6-27. (d)Debouck,C. T h e m - 1 (Whittaker BioProducts), 10% inactivated fetal bovine serum, Protease as a Therapeutic Target for AIDS. AIDS Res. Human 4 mM glutamine (Gibco Labs), and 1:lOOpenicillin/streptomycin Retroviruses 1992,8(2),153-64.(e)Martii, J.A. Recent Advan(Gibco Labs). Cells were treated with the compound for 24 h in the Design of HIV Protease Inhibitors. AntiuiralRee. 1992,17, 266-78. prior to HIV-1 infection. Cells were infected at day 0 at a (3)Roberta, N. A.; Martin,J. A,; Kinchington,D.; Broadhurst,A. V.; concentration of 250 OOO per mL with a 1:2OOO dilution of HIV-1 Craig, J. C.; Duncan, I. B.; Galpin, S. A.; Handa, B. K.;Kay, J.; variant. The multiplicityof infection was 0.01 in all cases. Fresh Krohn, A.; Lambert,R. W.; Merrett, J. H.; Mille, J. 9.; Parkes, K. compound was added at the time of infection and every 2-3 days E.B.;Redshaw, S.; Ritchie, A. J.; Taylor, D. L.; Thomae, G. J.; thereafter. Incubations were performed at 37 OC in a CO2 Machin, P. J. Rational Design of Peptide-Based HIV Proteinase Inhibitors. Science 1990,248,368-61. atmosphere. H9 and MT-4 human T-lymphoid cells are described (4)Thompson, W.J.; Ghoeh, A. K.;Holloway, M. K.;Lee, H. Y.; by Popovic et al.n and Miyoshi et al.,a respectively. Primary Munson, P. M.;Schwering, J. E.;Wai, J. M.;Darke, P. L.; Zugay, peripheral blood lymphocytes and primary monocytes/macrophJ. A.; Emini,E.A.; Schleif, W. A.; Huff, J. R.;Anderson, P. S. ages were obtained from fresh human plasmaphoresis residues. 3'-Tetrahydrofuranylglycine as a Novel, Unnatural Amino Acid The monocytes/macrophages were separated from the lymphoSurrogate for Asparagine in the Design of Inhibitors of the HIV Protease. J. Am. Chem. SOC.1993,115,801-03. cytes by adherence to plastic and were maintained in GM-CSF-
23101 Journal of Medicinal Chemistry,1993, Vol. 36, No. 16 (5) Gao, Y.; Hanson, R. M.; Klunder, J. M.; KO,S. Y.; Masamune, H.; Sharpless, K.B. Catalytic Asymmetric Epoxidation and Kinetic Resolution: Modified ProceduresIncluding in Situ Derivatization. J. Am. Chem. SOC. 1987,109, 5765-80. Caron, M.; Carlier, P. R.; Sharpless, K. B. Regiceelective Azide Opening.of 2,eEpoxy Alcohols by [Ti(O-i-Pr)2(Na)2]: Synthesis of a-Ammo Acida J. Org. Chem. 1988,53,5185-87. Ghoeh, A. K.; McKee, 5.P.; Lee,H. Y.; Thompson, W. J. A Facile and Enantiospecific Synthesis of 2(S)- and B(R)-[l’(S)-Azido-2phenylethylloxirane. J. Chem. SOC., Chem. Commun. 1992,27374. Taaden, V. K.; van Leueen, A. M.; Wynberg, H. Synthesis of Enantiomerically pure (5)-(+)-3-Hydroxytetr~y~ofuran and ita REnantiomer from Malic or Tartaric Acid J. Org.Chem. 1983,48, 2767-69. Zaugg, H. E.; Dunnigan, D. A.; Michaels, R. J.; Swett, L. R.; Wang, T. S.; Sommers, A. H.; DeNet, R. W. Specific Solvent Effecta in the Alkylation of Enolate Anions. 111. Preparative Alkylations in Dimethylformamide. J. Org. Chem. 1961,26,644-651. Touseaint, 0.;Capdevielle,P.; Maumy,M. The Copper (I)Catalyzed Decarboxylation of Malonic Acide: A New Mild and Quantitative Method. Synthesis 1986,1029-31. Evane, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. The Asymmetric Synthesisof a-Amino Acids. Electrophilic Azidation of Chiral Imide Enolatea, a Practical Approach to the Synthesis of (R)- and (S)-a-Azido Carboxylic Acids. J. Am. Chem. SOC.1990, 112,4011-30. (a) Cragoe, E. J.; Woltersdorf, 0. W.; Bicking, J. B.; Kwong, S. F.; Jones, J. H. Pyrazine Diuretics. 11. N-Amidino-3-amino-5-substituted-6 Halopyrazinecarboxamidee. J. Med. Chem. 1967, 10, 66-75. (b) Jones, J. H.; Holtz, W. H.; Cragoe, E. J. Pyrazine Diuretics. VII. N-Amidino-3-substituted Pyrazinecarboxamides. J. Med. Chem. 1969,12, 285-88. (a) Handa, B. K.; Machin, P. J.; Martin, J. A.; Redshaw, S.; Gareth, T. J. Eur. Pat. Appl. 0346847A2. (b) J. T. Martin, J. A.; IZedshaw, S. Eur. Pat. Appl. 0432695A2. (c) Hayashi, K.; Ozaki, Y.; Nunami, K.4.; Yoneda, N. Facile Preparation of Optically Pure (3s)-and (3R)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic Acid. Chem. Phurm. Bull. 1983,31,312-4. Small amounta of the diastereomers of 18 were eliminated by a single recrystallization from ethyl acetate.
Evane,B.E.;Rittle,K.E.;Homnick,C.F.;Springer,J.P.;Hirshfield,
J.: Veber, D. F. A Stereocontrolled Svnthesis of Hvdroxvethvlene Dipeptide h t e r e s Using Novel Chir-al, Aminoalkjrl EpdxidG and y-(Aminaalkyl) y-Ladones. J. Org. Chem. 1985,50,4615-25. Bernasconi, S.; Comini, A.; Corbella, A.; Gariboldi, P.; Sisti,M. Activation of 2-Alkenoic Acids as Mixed Anhydrides with Diphenylphosphinic Acid for the Formation of Carboxamides. Synthesis 1980,385-387. Priebe, W.; Grynkiewicz, G. A Facile and Selective Oxidation of Sulfides to Sulfones. Tetrahedron Lett. 1991,32,7353-56. Thompson, W. J.; Fitzgerald, P. M. D.; Holloway, M. K.; Emini, E. A.; Darke, P. L.; McKeever, B. M.; Schleif, W. A.; Quintero, J. C.; Zugay, J. A,; Tucker, T. J.; Schwering, J. E.; Homnick, C. F.; Nunberg, J.; Springer, J. P.; Huff, J. R. Synthesis and Antiviral Activityof aSeriesof HIV-1Protease InhibitorswithFunctionality Tethered to the PI or PI! Phenyl Substituents X-ray Crystal Structure Aesisted Design. J. Med. Chem. 1992,35,1685-01 and references cited therein. Halgren, T. A.; Merck Sharp and Dohme Research Laboratories, Rahway, NJ. Unpublished work on the development of the force field program OPTIMOL. OPTIMOL differs from MM2 mainly in the use of partial charges on atoms, instead of bond dipoles, and in the absence of unshared paire of electrons on certain nitrogen and oxygen atoms.
Ghosh et al. Allinger, N. L. Conformational Analysis. 130. MM2. A Hydrocarbon Force Field Utilizing V1 and V2 Torsional Terms. J. Am. Chem. Soc. 1977,99,8127-34. The Ro 31-8959was prepared from amine 20 and 2 equiv of BOCasparagine using 3,4-dihydro-3-hydroxy-4oxo-l,2,3-benzotriazine (Fluka #37305) and EDC as coupling agent in 1:l ethyl acetateCHZClZsolvents and Nfl-diiiopropylethylamine asbase. Removal of the BOC group with TFA-CHFlI followed by coupling with quinaldic acid as described above gave stereochemically pure compound in 74% overall yield. Heimbach, J. C.; Gmky, V. M.; Micheleon, S. R.; Dixon, R. A.; SigalJ. S.; Darke,P. L. Affiinity Purification ofthe HIV-1Protease. Biochem. Biophys. Res. Commun. 1989,164,955-60. In thie model the 0-N distance from the asp side chain oxygen of Ro 31-8959 to the asp 29 and asp 30 nitrogen atoms were 2.68 A and 3.11 A. The 3%-Thfg side chain oxygen atom of inhibitor 23 was 2.81 A and 3.21 A, respectively. Abraham, M. H.; Duce, P. P.; Prior, D. V.; Barratt, D. G.; Morris, J. J.; Taylor, P. J. Hydrogen Bonding. Part 9. Solute Proton Aceptor Scalea for Use in Drug Design. J. Chem. SOC.,Perkin Trans. 2 1989,1355-75. Unless otherwise indicated all determinations were n = 1. (a) The HIV-2 PR (ROD) was expreseed with the same system as the HIV-1 PR which was previously described; Darke, P. L.; Leu, C.-T.; Davia, L.; Heimbach, J. C.; Diehl, R. E.; Hill, W.5.;Dixon, R. A. F.; Sigal, I. S. Human Immunodeficiency Virus Protease: Bacterial Expreeeionand Characterization of the Purified Aspartic Protease. J. Biol. Chem. 1989,264,2307-12. (b) The HIV-2 PR (ROD)was purifed with the same methods as the HIV-1 PR which was previously described; see ref 22. Popovic,M.; Samgadharan, M. G.; Read, E.; Gallo,R. C. Detection, Isolation, and Continuous Production of Cytopathic Retroviruses (HTLV-III) frompatientawithAIDS andPre-AIDS. Science 1984, 224,497-500. Miyoshi, I.; Kubonishi, I.; Yoshimoto, S.; Akagi, T.; Ohtauki, Y.; Shiraishi, Y.; Nagata, K.; Hinuma, Y. Type C V i Particlee in a Cord T-cell Line Derived by Co-cultivating Normal Human Cord Leukocytes and Human Leukaemic T-Cella. Nature (London) 1981,2.94,770-71. AMF is an extension of work described previously: (a) Gund, P.; Andose, J. D.; Rhodes, J. B.; Smith, G. M. Three Dimensional Molecular Modeling and Drug Design. Science 1980,208,142531. (b) Smith, G. M.; Hangauer, D. G.; Andoee, J. D.; Bush, B. L.; Fluder, E. M.; Gund, P.; McIntyre, E. F. Intermolecular Modeling Methode in Drug Design: Modeling the Mechanism of Peptide Cleavage by Thermolysin. Drug Znf. J. 1984,18, 167-178. The major authors of the AMF program are J. D. Andose, R. A. Blevins, E. M. Fluder, and J. Shpungin. Giam, C.-Z.;Boros, LZn Viuo andZn VitroAutoproceasingofHuman Immunodeficiency Virue Protease Expressed in Escherichia coli. J. Biol. Chem. 1988,263, 14617-20. This ie based on the pH-rate profile for fungal aspartyl protease (Hofmann, T.; Hodges, 5.R.; James, M. N.G. Effect of pH on the Activities of Penicillopepsin and Rhizopus Pepsin and a Propoeal for the Productive Substrate Binding Mode in Penicillopepin. Biochemistry 1984,23,635-43)whichsuggestathat thetwo catalytic aspartic acida share one proton in the active pH range. Commercially available software from Polygen Corporation 200 Fifth Avenue, Waltham, MA 02245.
